结核分枝杆菌北京家族菌株的起源、进化及其与现代人类迁移的联系
发布时间：2019-06-25 11:47
【摘要】：我国是全球22个结核高负担国家之一,结核病人数位居世界第二。我国的结核疫情主要由结核分枝杆菌北京家族菌株(简称北京菌株)所致。该菌株在东亚各国高度流行,并在全球广泛传播。在表型上,北京菌株通常被认为具有更强的致病性和耐药性,但不同实验或地区的结果并不一致；在基因型上,北京菌株被认为是一个遗传相似度极高的群体,但近期大量分型或测序数据提示该群体内部存在一定的遗传差异性。目前为止,北京菌株的遗传结构仍缺乏系统的研究；北京菌株的起源问题也存在争议。了解北京菌株的起源、进化和传播历史对进一步认识其在全球的广泛分布及其致病性和耐药性有重要意义。 通过对从全球范围内收集的58株北京菌株的22个3R基因进行测序分析,我们首先建立了北京菌株高分辨率的系统发生树；根据该系统树,北京菌株可被进一步分为多个相互间具有一定遗传距离的亚型。随后通过对305株北京菌株进行单核苷酸多态性(SNP)分型,我们进一步研究了全球北京菌株的群体结构。分型结果提示,全球范围流行的北京菌株主要由一个近期进化出的Bmyc10亚型所致。 为了进一步探索北京菌株的起源、进化及其与人类迁移的联系,我们对全国范围内11个省市收集的北京菌株进行了SNP分型。同时我们从上海市收集的北京菌株中挑选了各亚型的代表菌株(共计39株)进行全基因组测序。联合已发表的22个结核全基因组测序数据,我们构建了全基因组系统发生树；同时,通过贝叶斯进化分析我们推算了北京及其亚型菌株的起源年代。根据该系统发生树,我们发现前期在东南亚地区分离的“前北京”菌株代表了北京菌株祖先最早分化的一个分枝；而对全国范围内的菌株分型结果表明“前北京”菌株非常稀少且仅存在于我国南方地区(广西、福建、浙江和四川)。时间推算结果显示北京菌株的起源年代为距今2-3万年左右,该时间和东亚人口在东南亚起源的年代完全相符。综上我们推断北京菌株很可能起源于东南亚和我国南方交界的地区。 北京家族各亚型菌株在11个省市的分布表明,早期分化的“古老”型菌株在全国广泛存在,且在西藏和日韩等地为主要流行菌株。联合东亚早期人口迁移路线,我们推测北京菌株在东南亚一带起源后,随着人群从南向北的迁移传播到我国大部分地区并到达日韩等地。“现代”型菌株是我国除西藏外其他地区的主要流行菌株,其在汉族人群中的比例显著高于在少数名族人群中的比例。而汉族人群中,北方人群中的比例显著高于南方。因此我们推测“现代”型菌株可能起源于北方汉族的祖先人群中。全基因组系统发生树显示“现代”型菌株在距今约6000年前发牛了次大规模的扩张,这与“新石器”时期农业产生后仰韶社会人群(北方汉族祖先)扩张的年代完全相符,从而进一步证明了“现代”菌株很可能起源于我国北方。北方汉族人群在近2000多年的时间在东亚发生了大规模扩张和迁移,我们推测“现代”型菌株在我国及东亚各地区广泛的分布很可能与汉族人群的迁移相关。在汉族人口较少的西藏和日韩等地,“现代”型菌株比例甚至低于“古老”型,从而进一步证明了这一假设。 “现代”型北京菌株在早期北方人群中的扩张及后来在东亚及世界各地广泛流行都提示该亚型菌株很可能具有较强的致病性。我们相信通过更深入的比较基因组学研究很可能找出该亚型菌株菌株成为优势菌株的遗传学机制。另外,“现代”菌株在我国的高度流行提示结防部门需加大病例发现和早期诊断的力度,从而降低该菌株在人群中的传播和进一步扩张。 “现代”型北京菌株在我国庞大群体数量及菌株间高度的遗传相似性使得研究该菌株的近期传播比较困难。目前欧美推行的15和24位点可变串联重复序列(VNTR)分型方法在运用于我国流行的北京菌株群体时往往存在簇病例间无流行病学联系的情况。通过对上海市崇明地区以人群为基础收集的191株北京菌株进行15位点VNTR和8位点SNP分型,我们发现5个15位点定义的簇菌株包含了两个或多个北京亚型的菌株。通过联合VNTR和SNP构建系统树,我们证明以上现象一方面是由于15位点VNTR对“现代”型菌株分辨力偏低所致；另一方面,“现代”型菌株间通过趋同或平行进化形成的VNTR基因型非同源相似性也会导致错误的定义簇菌株。所以,我们推荐联合SNP和VNTR分型在“现代”型北京菌株高度流行的地区进行分子流行病学研究。但目前为止,仍缺乏统一的VNTR分型方案用于我国结核分子流行病学研究。标准的15和24位点VNTR因其位点数较多且很多位点分辨力较低而不太适用于我国结核分型。通过对筛选的24个位点在全国范围6个地区以人群为基础收集的1375株菌株中进行分辨力及各种VNTR组合的HGI值进行研究,我们挑选了广泛适用的10个VNTR位点(MIRU10,26,31,40; QUB11b,18,26; Mtub04,21及ETR-A)作为研究我国结核分子流行病学的一线分型方案。这10个位点联合3个高变位点及QUB11a作为二线分型方案,可准确的定义不同地区菌株的近期传播。 全基因组比对的结果表明,北京各亚型菌株间存在一定的遗传学差异。我们推测这些差异会导致不同亚型菌株间对抗生素适应性的异同。通过对上海市疾控中心2004-2008年收集的242个耐多药菌株进行SNP分型及一线药物耐药突变分析,我们发现高度流行的“现代”型北京菌株在rpoB531位点的突变频率较其在其它亚型菌株高(OR=1.8,P=0.04)；而在几个低度流行的“古老”亚型菌株中,利福平耐药突变主要分布在rpoB531和rpoB526位点以外(OR=3.16,P0.01)。rpoB531和ropB526位点的突变由于对细菌适应力的损伤较低,被认为是导致利福平耐药的优势突变。“现代”型菌株可能具有更强的致病性使其在病人体内具有更高的菌载量,从而更易筛选出携带优势突变的耐药菌株。根据这242个耐多药菌株的突变特征,我们筛选出了11个位点用于预测一线药物耐药表型。该11位点可预测93.8%利福平耐药,89.3%异烟肼耐药及86.8%耐多药表型。基于这些突变位点,我们进一步研发了一种基于实时荧光PCR探针溶解曲线分析的新技术用于耐药结核的快速诊断。由于该方法只用了一种荧光标记的探针,所以成本较低且可广泛适用于各种荧光PCR检测平台。 综上,本论文通过研究结核北京菌株的起源和进化进一步揭示了结核的传播与现代人类迁移、扩张之间密切的联系。我们的结论为进一步研究结核菌致病机制提供了重要线索。同时我们为我国结核分子流行病学研究提供了可靠的、广泛适用的分型方案；为耐药结核的快速诊断提供了可靠的分子生物学方法。
[Abstract]:China is one of the 22 high-burden countries in the world, and the number of tuberculosis is the second in the world. The tuberculosis epidemic in China is mainly caused by the Beijing family strain of Mycobacterium tuberculosis (the Beijing strain). The strain is highly popular in the East Asian countries and is widely disseminated in the world. In the phenotype, the Beijing strain is generally considered to have stronger pathogenicity and drug resistance, but the results of different experiments or regions are not consistent; in the genotype, the Beijing strain is considered to be a highly genetic population, However, in recent years, a lot of typing or sequencing data suggest that there is a certain genetic difference within the population. So far, the genetic structure of the Beijing strain still lacks a systematic study, and the origin of the Beijing strain is also controversial. To understand the origin, evolution and propagation history of the Beijing strain, it is of great significance to further know its wide distribution and its pathogenicity and drug resistance in the world. By sequencing and analyzing the 22 3R genes of 58 Beijing strains collected worldwide, we first set up a high-resolution system-generating tree of Beijing strain. According to the system tree, the Beijing strain can be further divided into a plurality of sub-regions with a certain genetic distance from each other. Then, by using the single nucleotide polymorphism (SNP) of 305 Beijing strains, we further studied the group knot of the global Beijing strain. The results suggest that the global-wide Beijing strain is mainly composed of a recently-developed Bmyc10 subtype. To further explore the origin and evolution of the Beijing strain and its relation to human migration, we have carried out the SN of the Beijing strain collected in 11 provinces and cities throughout the country. P. At the same time, we selected the representative strains of each subtype (total of 39 strains) from the Beijing strain collected in Shanghai to carry out the whole gene. Group sequencing. The whole genome sequencing data of 22 nodules that have been published, we constructed the whole genome phylogenetic tree; at the same time, through the Bayesian evolution analysis, we calculated the origin of the Beijing and its subtype strains In the source age, according to the phylogenetic tree, we found that the "Former Beijing" strains isolated in the early stage represent the first branch of the earliest differentiation of the ancestors of the Beijing strain; and the typing of the strains in the whole country shows that the "Former Beijing" strains are very rare and only exist in the southern region of China (Guangxi, Fujian, Zhejiang and The time-reckoning result shows that the origin of the Beijing strain is about 2-30,000 years, and the time and the East Asian population are over the period of the origin of South-East Asia. Overall, we conclude that the Beijing strain is likely to originate from South-East Asia and the southern border of our country The distribution of each subtype of the Beijing family in 11 provinces shows that the early-differentiated "ancient"-type strain is widely present in the whole country, and it is mainly in Tibet and Japan in combination with that early population migration route in east Asia, we have speculated that the Beijing strain, after the origin of the south-east Asia region, has spread to most of our region with the migration of the population from south to north, The "modern"-type strain is the main epidemic strain of China except Tibet, the proportion of which is significantly higher in the Han population than in the minority. The proportion of the population in the population, and the proportion of the northern population in the Han population It's higher than the South. So we're speculating that the "modern"-type strain may have originated in the northern Han population The phylogenetic tree of the whole genome shows that the "modern"-type strain has been expanded on a large scale from about 6000 years ago, which is the same as that of the back-up social group (the ancestors of the northern Han people) after the agricultural production in the "Neolithic" period. The generation of the "modern" strain is further proved to be of the origin In the northern part of China, the large-scale expansion and migration of the Chinese Han population in East Asia over the last 2000 years, we have speculated that the modern "-type strain is widely distributed in the regions of China and East Asia and is likely to be the same as that of the Han population The proportion of the "modern"-type strain is even lower than that of the "ancient" type in the Tibetan and Japanese-Korean and other places in the Han population. This assumption is that the expansion of the "modern"-type Beijing strain in the early population of the North and later in East Asia and the wide spread of the world suggest that the subtype strain is likely to be It is highly pathogenic. We believe that by a more in-depth comparative genomics study, it is possible to identify the strain of this subtype as an advantage. In addition, the high prevalence of the "modern" strain in China suggests that the prevention and control department need to increase the number of case discovery and early diagnosis, so as to reduce the transmission of the strain in the population The genetic similarity of "modern"-type Beijing strain on the number of large population and the height of the strain in China, so as to study the strain The recent communication between the 15 and 24-site variable tandem repeats (VNTR) in Europe and the United States has been applied to the popular Beijing strain population in China, and there is often no cluster case among the cluster cases. According to the epidemiological connection, we found that five 15-site-defined cluster strains contained two or more of the 191 Beijing strains collected on the basis of the population-based population in Chongming, Shanghai. A number of strains of the Beijing Subtype. The system tree was constructed by the combination of VNTR and SNP, and we prove that the above phenomenon is due to the 15-site VNTR's resolution of the "modern"-type strain On the other hand, the non-homologous similarity of the VNTR genotypes formed by the convergent or parallel evolution between the "modern"-type strains can also guide The wrong definition of cluster strains. Therefore, we recommend the combination of SNP and VNTR typing in the highly popular region of the "modern"-type Beijing strain To date, there is still a lack of a unified VNTR classification scheme for the junction of our country. Nuclear molecular epidemiological studies. The standard 15 and 24 sites, VNTR, are not too numerous and have a low resolution of many sites We selected a wide range of 10 VNTR sites (MIRU10,26,31,40; QUB) by conducting a study on the resolution of the screened 24 sites in a population-based 1375 strain and a variety of VNTR-combined HGI values in a population-based collection of 1375 strains in six regions of the country (11b,18,26; Mtb04,21 and ETR-A) as a study of the prevalence of tuberculosis in China The first line of the disease is a first-line classification scheme. The 10 sites are combined with 3 high-displacement points and QUB11a as the two-line classification scheme, and the exact definition is different. Short-term spread of the regional strains. The results of the full-genome comparison show that all the strains of Beijing There is a certain genetic difference between them. We are speculating that these differences can lead to different subtype strains We found that the mutation frequency of the high-prevalence "modern"-type Beijing strain at the rpoB531 site is higher than that of other subtype strains (OR =) by using the SNP typing and the first-line drug-resistance mutation analysis of 242 drug-resistant strains collected in the disease control center in Shanghai in 2004-2008. 1.8, P = 0.04); whereas in several low-prevalence "ancient" subtype strains, rifampin-resistant mutations were mainly distributed outside the rpoB531 and rpoB526 sites (OR = 3.16, P0.01). The mutation at the rpoB531 and pB526 sites is considered to be a guide due to the low damage to the bacterial strain The "modern"-type strain may have stronger pathogenicity so that it has higher bacterial load in the patient and is more easily screened out. The resistance strain carrying the dominant mutation is screened out for 11 sites according to the mutation characteristics of the 242 multi-drug resistant strains. The first-line drug-resistant phenotype was predicted. The 11-site could be predicted to be 93.8% rifampin resistance and 89.3% of the drug-resistant drug resistance. and 86.8% of the multi-drug resistant phenotype. Based on these mutation sites, we further developed a new technology based on real-time fluorescence PCR probe dissolution profile analysis The method has the advantages of low cost and wide application to the rapid diagnosis of drug-resistant tuberculosis, In this paper, we study the origin and evolution of the Beijing strain of the tuberculosis, and further reveal the transmission of the tuberculosis and the modern people. A close link between the migration and expansion of the class. Our conclusion is to further study the knot. At the same time, we provide a reliable and widely applicable classification scheme for the epidemiological study of tuberculosis in our country, and provide a rapid diagnosis for drug-resistant tuberculosis.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R378.911
本文编号：2505659
[Abstract]:China is one of the 22 high-burden countries in the world, and the number of tuberculosis is the second in the world. The tuberculosis epidemic in China is mainly caused by the Beijing family strain of Mycobacterium tuberculosis (the Beijing strain). The strain is highly popular in the East Asian countries and is widely disseminated in the world. In the phenotype, the Beijing strain is generally considered to have stronger pathogenicity and drug resistance, but the results of different experiments or regions are not consistent; in the genotype, the Beijing strain is considered to be a highly genetic population, However, in recent years, a lot of typing or sequencing data suggest that there is a certain genetic difference within the population. So far, the genetic structure of the Beijing strain still lacks a systematic study, and the origin of the Beijing strain is also controversial. To understand the origin, evolution and propagation history of the Beijing strain, it is of great significance to further know its wide distribution and its pathogenicity and drug resistance in the world. By sequencing and analyzing the 22 3R genes of 58 Beijing strains collected worldwide, we first set up a high-resolution system-generating tree of Beijing strain. According to the system tree, the Beijing strain can be further divided into a plurality of sub-regions with a certain genetic distance from each other. Then, by using the single nucleotide polymorphism (SNP) of 305 Beijing strains, we further studied the group knot of the global Beijing strain. The results suggest that the global-wide Beijing strain is mainly composed of a recently-developed Bmyc10 subtype. To further explore the origin and evolution of the Beijing strain and its relation to human migration, we have carried out the SN of the Beijing strain collected in 11 provinces and cities throughout the country. P. At the same time, we selected the representative strains of each subtype (total of 39 strains) from the Beijing strain collected in Shanghai to carry out the whole gene. Group sequencing. The whole genome sequencing data of 22 nodules that have been published, we constructed the whole genome phylogenetic tree; at the same time, through the Bayesian evolution analysis, we calculated the origin of the Beijing and its subtype strains In the source age, according to the phylogenetic tree, we found that the "Former Beijing" strains isolated in the early stage represent the first branch of the earliest differentiation of the ancestors of the Beijing strain; and the typing of the strains in the whole country shows that the "Former Beijing" strains are very rare and only exist in the southern region of China (Guangxi, Fujian, Zhejiang and The time-reckoning result shows that the origin of the Beijing strain is about 2-30,000 years, and the time and the East Asian population are over the period of the origin of South-East Asia. Overall, we conclude that the Beijing strain is likely to originate from South-East Asia and the southern border of our country The distribution of each subtype of the Beijing family in 11 provinces shows that the early-differentiated "ancient"-type strain is widely present in the whole country, and it is mainly in Tibet and Japan in combination with that early population migration route in east Asia, we have speculated that the Beijing strain, after the origin of the south-east Asia region, has spread to most of our region with the migration of the population from south to north, The "modern"-type strain is the main epidemic strain of China except Tibet, the proportion of which is significantly higher in the Han population than in the minority. The proportion of the population in the population, and the proportion of the northern population in the Han population It's higher than the South. So we're speculating that the "modern"-type strain may have originated in the northern Han population The phylogenetic tree of the whole genome shows that the "modern"-type strain has been expanded on a large scale from about 6000 years ago, which is the same as that of the back-up social group (the ancestors of the northern Han people) after the agricultural production in the "Neolithic" period. The generation of the "modern" strain is further proved to be of the origin In the northern part of China, the large-scale expansion and migration of the Chinese Han population in East Asia over the last 2000 years, we have speculated that the modern "-type strain is widely distributed in the regions of China and East Asia and is likely to be the same as that of the Han population The proportion of the "modern"-type strain is even lower than that of the "ancient" type in the Tibetan and Japanese-Korean and other places in the Han population. This assumption is that the expansion of the "modern"-type Beijing strain in the early population of the North and later in East Asia and the wide spread of the world suggest that the subtype strain is likely to be It is highly pathogenic. We believe that by a more in-depth comparative genomics study, it is possible to identify the strain of this subtype as an advantage. In addition, the high prevalence of the "modern" strain in China suggests that the prevention and control department need to increase the number of case discovery and early diagnosis, so as to reduce the transmission of the strain in the population The genetic similarity of "modern"-type Beijing strain on the number of large population and the height of the strain in China, so as to study the strain The recent communication between the 15 and 24-site variable tandem repeats (VNTR) in Europe and the United States has been applied to the popular Beijing strain population in China, and there is often no cluster case among the cluster cases. According to the epidemiological connection, we found that five 15-site-defined cluster strains contained two or more of the 191 Beijing strains collected on the basis of the population-based population in Chongming, Shanghai. A number of strains of the Beijing Subtype. The system tree was constructed by the combination of VNTR and SNP, and we prove that the above phenomenon is due to the 15-site VNTR's resolution of the "modern"-type strain On the other hand, the non-homologous similarity of the VNTR genotypes formed by the convergent or parallel evolution between the "modern"-type strains can also guide The wrong definition of cluster strains. Therefore, we recommend the combination of SNP and VNTR typing in the highly popular region of the "modern"-type Beijing strain To date, there is still a lack of a unified VNTR classification scheme for the junction of our country. Nuclear molecular epidemiological studies. The standard 15 and 24 sites, VNTR, are not too numerous and have a low resolution of many sites We selected a wide range of 10 VNTR sites (MIRU10,26,31,40; QUB) by conducting a study on the resolution of the screened 24 sites in a population-based 1375 strain and a variety of VNTR-combined HGI values in a population-based collection of 1375 strains in six regions of the country (11b,18,26; Mtb04,21 and ETR-A) as a study of the prevalence of tuberculosis in China The first line of the disease is a first-line classification scheme. The 10 sites are combined with 3 high-displacement points and QUB11a as the two-line classification scheme, and the exact definition is different. Short-term spread of the regional strains. The results of the full-genome comparison show that all the strains of Beijing There is a certain genetic difference between them. We are speculating that these differences can lead to different subtype strains We found that the mutation frequency of the high-prevalence "modern"-type Beijing strain at the rpoB531 site is higher than that of other subtype strains (OR =) by using the SNP typing and the first-line drug-resistance mutation analysis of 242 drug-resistant strains collected in the disease control center in Shanghai in 2004-2008. 1.8, P = 0.04); whereas in several low-prevalence "ancient" subtype strains, rifampin-resistant mutations were mainly distributed outside the rpoB531 and rpoB526 sites (OR = 3.16, P0.01). The mutation at the rpoB531 and pB526 sites is considered to be a guide due to the low damage to the bacterial strain The "modern"-type strain may have stronger pathogenicity so that it has higher bacterial load in the patient and is more easily screened out. The resistance strain carrying the dominant mutation is screened out for 11 sites according to the mutation characteristics of the 242 multi-drug resistant strains. The first-line drug-resistant phenotype was predicted. The 11-site could be predicted to be 93.8% rifampin resistance and 89.3% of the drug-resistant drug resistance. and 86.8% of the multi-drug resistant phenotype. Based on these mutation sites, we further developed a new technology based on real-time fluorescence PCR probe dissolution profile analysis The method has the advantages of low cost and wide application to the rapid diagnosis of drug-resistant tuberculosis, In this paper, we study the origin and evolution of the Beijing strain of the tuberculosis, and further reveal the transmission of the tuberculosis and the modern people. A close link between the migration and expansion of the class. Our conclusion is to further study the knot. At the same time, we provide a reliable and widely applicable classification scheme for the epidemiological study of tuberculosis in our country, and provide a rapid diagnosis for drug-resistant tuberculosis.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R378.911
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